1. Uridylation of miRNAs by HEN1 SUPPRESSOR1 in Arabidopsis
Guodong Ren, Xuemei Chen, Bin Yu 
Current Biology 
Volume 22, Issue 8, Pages (April 2012)
DOI: /j.cub
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2. Figure 1
heso1-2 Mutation Partially Suppresses the Phenotypes of hen1-1 and hen1-2
(A) Morphological phenotypes of the indicated genotypes.
(B) Siliques from plants of the indicated genotypes. Ler is WT plants. hen1-1 heso1-2 and hen1-2 heso1-2 are hen1-1 and hen1-2 harboring the heso1-2 mutation, respectively. Comp is hen1-2 heso1-2 harboring the HESO1 genomic DNA.
(C) Average silique length in various genotypes. Thirty siliques from six plants for each genotype were included in the analysis. ∗∗∗p <
(D) The transcript levels of miRNA and ta-siRNA targets in various genotypes. Target mRNA accumulation in various genotypes was quantified by qRT-PCR and compared with those of WT. Quantifications are normalized with ACTIN2 transcript. UBQ5: UBQUITIN5, which served as an internal control. The WT value is 1. ∗p < 0.05; ∗∗p <0.01; ∗∗∗p <
Current Biology  , DOI: ( /j.cub )
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3. Figure 2 heso1-2 Affects miRNA and siRNA Profiles in hen1-1 and hen1-2
miRNAs and siRNAs in various genotypes were monitored by northern blot. U6 was used as a loading control. Ler served as a size control. miR166/165 and miR171/170, the paired miRNAs were recognized by the same probe because of sequence similarities.
The number below hen1-1 heso1-2 and hen1-2 heso1-2 indicated the fold changes of total amounts of miRNAs or siRNAs caused by heso1-2 in hen1-1 and hen1-2, respectively. n.d. represents not detected in hen1-1. To obtain the total amount of a miRNA and siRNA in each genotype, we quantified all individual bands in each sample and added them together. The total amount of a miRNA and siRNA in hen1-1 heso1-2 and hen1-2 heso1-2 was then normalized to U6 RNA and compared with that in hen1-1 and hen1-2, respectively.
Current Biology  , DOI: ( /j.cub )
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4. Figure 3 HESO1 Encodes a 3′ Terminal Uridylyl Transferase
(A) Schematic of HESO1 protein and its mutant forms generated by heso1-2. ∗ represents protein stop codon. The amino acids introduced by heso1-2 following the truncated HESO1 are indicated. PAP/25A, poly A polymerase domain; PAPA, PAP associated domain; Core, core regions of PAP/25A; GLN_RICH, glutamine rich region.
(B) Terminal uridylyl transferase activity of HESO1. Oligo 248 was 5′-end labeled with P32, incubated with buffer alone (1), purified MBP (2), MBP-HESO1 (3) or MBP-HESO1Δ16 (4) under the presence of UTP for 120 min, and resolved on a denaturing polyacrylamide gel.
(C) Time-course reaction of HESO1 activity and the effect of 2′-O-methylation on HESO1 activity. 5′ end–labeled oligo 248 or 2′-O-methyl oligo 248 was incubated with MBP-HESO1. The reaction was stopped at the indicated time.
(D) Preference of HESO1 to ribonucleotides and deoxyribonucleotides. 5′ end labeled oligo 248 was incubated with MBP-HESO1 under the presence of UTP, ATP, CTP, GTP, dATP, dTTP, dCTP, or dGTP for 40 min.
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5. Figure 4
Overexpression of HESO1 in hen1-2 Causes More Severe Phenotypes
(A) Morphological phenotypes of various genotypes. Ler is WT plants. hen1-2 + 35S::HESO1 is hen1-2 overexpressing HESO1.
(B) miR167 and miR166/165 in various genotypes were monitored by northern blot. U6 was used as a loading control. Ler served as a miRNA size control; hen1-2 + 35S::HESO1, hen1-2 overexpressing HESO1. The number below overexpression lines indicated the fold changes of total amounts of miRNAs or siRNAs caused by overexpression of HESO1 in hen1-2, respectively. To obtain the total amount of a miRNA and siRNA in each genotype, we quantified all individual bands in each sample and added them together. The total amount of a miRNA and siRNA in HESO1 overexpression lines 2 was then normalized to U6 RNA and compared with that in hen1-2.
(C) The transcript levels of three miRNA targets in Ler, hen1-2, and three HESO1 overexpression lines. Target mRNA accumulation in various genotypes was quantified by qRT-PCR and compared with those of WT. Quantifications are normalized with ACTIN2 transcript. The hen1-2 value is 1. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <
Current Biology  , DOI: ( /j.cub )
Copyright © 2012 Elsevier Ltd Terms and Conditions